1Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada. katia.margiotti@vch.ca

Abstract

BACKGROUND:

The androgen receptor is a ligand-induced transcriptional factor, which plays an important role in normal development of the prostate as well as in the progression of prostate cancer to a hormone refractory state. We previously reported the identification of a novel AR coactivator protein, L-dopa decarboxylase (DDC), which can act at the cytoplasmic level to enhance AR activity. We have also shown that DDC is a neuroendocrine (NE) marker of prostate cancer and that its expression is increased after hormone-ablation therapy and progression to androgen independence. In the present study, we generated tetracycline-inducible LNCaP-DDC prostate cancer stable cells to identify DDC downstream target genes by oligonucleotide microarray analysis.

RESULTS:

Comparison of induced DDC overexpressing cells versus non-induced control cell lines revealed a number of changes in the expression of androgen-regulated transcripts encoding proteins with a variety of molecular functions, including signal transduction, binding and catalytic activities. There were a total of 35 differentially expressed genes, 25 up-regulated and 10 down-regulated, in the DDC overexpressing cell line. In particular, we found a well-known androgen induced gene, TMEPAI, which wasup-regulated in DDC overexpressing cells, supporting its known co-activation function. In addition, DDC also further augmented the transcriptional repression function of AR for a subset of androgen-repressed genes. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time RT-PCR.

CONCLUSION:

Taken together, our results provide evidence for linking DDC action with AR signaling, which may be important for orchestrating molecular changes responsible for prostate cancer progression.

LNCaP-DDC cell line displays regulated DDC expression with Dox treatment. Shown are the results from (A) Western blot and (B) semiquantitative RT-PCR analysis of the expression of DDC protein and mRNA, respectively. In A, LNCaP-DDC and LNCaP-pDEST (vector control) cells were treated for 48 hours under mock-induced (-Dox) and Dox-induced (+Dox) conditions before protein lysate preparation. No visible expression was detected in the LNCaP-pDEST control cells regardless of Dox treatment status (lines 3–4). A 6 fold increased DDC expression level was detected in the Dox-induced LNCaP-DDC cells compared to the mock-induced cells (lines 1–2), after β-actin normalization. In B, total cellular RNA was isolated from LNCaP-DDC and LNCaP-pDEST cells after 48 hours of Dox treatment. After reverse transcription, PCR was performed with DDC and β-actin specific primers. The up-regulation of the 209-bp DDC-specific band was detected in LNCaP-DDC cells (line 2). A 100 bp DNA ladder (Promega) was used for size markers.

Microarray analysis of DDC-regulated genes. Experimental outline of microarray studies to identify DDC downstream targets using LNCaP-DDC stable and control LNCaP-pDEST cells. In the presence of Dox (grey triangle, + Dox) the tetracycline Tet repressor (tetR) is released from the TetO2 sequence in the promoter of the lentiviral construct containing the DDC gene. The dissociation of the tetR allows induction of transcription for the gene of interest. LNCaP-DDC and LNCaP-pDEST (vector control) cell lines were plated in medium containing 5% charcoal-stripped serum and incubated overnight. The next day, cells were treated for 48 hours under mock-induced (-Dox) and Dox-induced (+Dox) conditions, 24 h of which was in the presence or absence of 0.1 nM R1881. Total RNA samples were isolated from each condition, labeled and hybridized on the microarrays. The relative mRNA abundance of each gene was calculated as a ratio between hormone-treated (+R1881) and hormone-untreated (- R1881) samples. The comparison of the expression data obtained from LNCaP-DDC+Dox stable cells (left) with the expression data from the three LNCaP control cells (right: LNCaP-DDC-Dox, LNCaP-pDEST- Dox, and LNCaP-pDEST+Dox) yielded the identification of genes that are androgen- and DDC-regulated.

Scatter plot of entire gene set considered expressed (p values = 0.05) in the microarray analysis. The position of each dot on the scatter plot corresponds to the normalized average signal intensity (log scale) of a single gene. The normalized average signal intensity under the DDC overexpression conditions are shown on the x and y axes (controls = no DDC overexpression and DDC = DDC overexpression). The middle line indicates values that represent a DDC/controls ratio of 1.0 (similar levels of expression in both cell lines). The outer lines represent a DDC/controls ratio of 2.0 (upper line; 2-fold greater expression in DDC compared to controls) and of 0.5 (lower line; 2-fold greater expression in controls compared to DDC).

Androgen-regulated and DDC-regulated genes. A) A Venn diagram analysis showing in yellow, the genes (130) with two fold induction in response to R1881 treatment in DDC overexpressing and control cells. The genes up- and down-regulated only in DDC overexpressing cells are represented in red (DDC; left) and the genes up- and down-regulated only in the control cells are represented in the green (controls; right). B) Reported here are 35 androgen-regulated genes differentially expressed at least 2-fold in DDC overexpressing cells (LNCaP-DDC) compared with the controls cells (LNCaP-CTRLs). The colors represent the ratio of gene expression levels in each cell line after R1881 treatment (red = hormone up-regulated and green = hormone down-regulated).